O objetivo desta Tese foi analisar os efeitos do treino de marcha isolado e em combinação com transplante de glia embainhante olfatória (GEO) na recuperação funcional e na plasticidade neuromuscular dependente da atividade em um modelo experimental de paraplegia. Para tanto, foram realizados 2 experimentos. No 1º experimento foi realizada completa transecção da medula espinal (TME) em ratos Wistar adultos e após 5 dias iniciou-se um protocolo de 9 semanas de treino de marcha em esteira com suporte de peso corporal. No 2º experimento, os animais receberam, imediatamente após a TME, transplante de células gliais embainhantes olfatórias (GEO) e, como no primeiro experimento, iniciaram o treino de marcha 5 dias após a lesão/transplante. Durante o período dos experimentos, estudos comportamentais para acompanhamento da recuperação da função sensório-motora dos animais foram periodicamente realizados. Além disso, ao término da fase de treinamento (10 semanas após a lesão/transplante), análises histológicas e bioquímicas foram realizadas em amostras de tecido retiradas da medula espinal e músculo sóleo. Os resultados mostram que o treino de marcha em esteira promove melhora da função sensório-motora nos membros posteriores (MPs) de ratos com completa transecção da medula espinal (TME). Os animais treinados apresentaram escores mais altos na escala BBB e normalização do reflexo flexor de retirada. Além disso, os animais com TME apresentaram atrofia do soma celular nos motoneurônios alfa, redução na expressão de sinaptofisina e na atividade da Na+,K+-ATPase na região lombar. Os animais treinados mostraram soma motoneuronal, expressão de sinaptofisina e atividade da bomba de Na+,K+-ATPase similares aos controles. No músculo sóleo, a TME causou severa atrofia muscular, que foi acompanhada pela redução na expressão do fator neurotrófico derivado do encéfalo (BDNF) neste músculo. Por outro lado, o treino de marcha foi capaz de parcialmente impedir/reverter a atrofia provocada pela paralisia muscular e promover um significante aumento na expressão do BDNF, o qual teve positiva correlação com o trofismo muscular dependente da atividade motora no músculo sóleo. O transplante de glia embainhante olfatória (GEO) promoveu significativo aumento nos escores da escala BBB nos animais com completa TME. Entretanto, o treino de marcha foi capaz de acelerar este ganho funcional. Apesar de não ser observada significativa regeneração axonal através do local da lesão, sugerindo que as melhoras funcionais ocorreram independentemente da existência de regeneração axonal. Estes resultados sugerem que o treino de marcha após a TME promove plasticidade morfológica e bioquímica dependente da atividade nos tecidos neuromusculares. A melhora funcional ocorreu concomitantemente a estas alterações plásticas. Além disso, a terapia de transplante de GEO mostrou resultados positivos na recuperação da função motora dos MPs que foi acelerada pelo treino de marcha, mesmo na ausência de regeneração axonal através da lesão. Estes dados mostram importantes informações neurobiológicas que fornecem base neurocientífica para o uso seguro e eficaz destas terapias na reabilitação após LME. / The aim of this thesis was to study the effects of treadmill step training alone and in combination with olfactory ensheathing cells (OEC) on functional recovery and activity-dependent neuromuscular plasticity in a traumatic paraplegia model. For this, we made two experiments. In the 1st experiment, complete spinal cord transection (SCT) was made in adult Wistar rats and after 5 days the spinal animals were underwent a 9 week body-weight-supported treadmill training (BWSTT) program. In the 2nd experiment, the spinal animals received acute olfactory ensheathing cell (OEC) transplantation and, similar to the 1st experiment, started a BWSTT 5 days after the injury/transplantation. Behavioral tests were periodically performed in order to study the hindlimb sensorimotor functions in both experiments. Furthermore, after 9 weeks of the training (10 weeks after SCI/transplantation), histological and biochemical analysis were performed in spinal cord and soleus muscle tissues. The results show that treadmill step training improves hindlimb sensorimotor function in rats with complete spinal cord transection (SCT). The trained animals showed higher BBB scores and normalization of the withdrawal reflex. Furthermore, spinal animals showed alpha motoneuron soma size atrophy, decrease in synaptophysin expression and Na+,K+-ATPase activity in lumbar spinal cord. Trained SCT animals showed motoneuron soma size, synaptophysin expression and Na+,K+-ATPase activity values similar to controls. In soleus muscle, SCT led to severe muscular atrophy, which was accompanied by a decrease in brain-derived neurotrophic factor (BDNF) expression in this muscle. On the other hand, treadmill step training was able to revert/prevent this paralysis-induced muscular atrophy and promote significant improvement in soleus BDNF expression, which was positively correlated to activity-dependent muscular trophism. Olfactory ensheathing cell (OEC) transplantation promotes significant improvements in the BBB scores of animals with SCT. However, treadmill step training was able to accelerate this functional gain. There was no significant axonal regeneration that traversed the injury site, which suggests that functional gains occurred in a manner independent of axonal regeneration. Taken as a whole, these results suggest that treadmill step training after SCT promotes activity-dependent morphological and biochemical plasticity in neuromuscular tissues. The functional improvements occurred concomitantly to these plastic changes. Moreover, OEC therapy showed positive results on hindlimb motor function recovery which was accelerated with treadmill step training even in the absence of axonal regeneration across the lesion site. These results represent important neurobiological information for the neuroscientific basis that supports these therapies as an efficient and safe approach in spinal cord injury rehabilitation.

Les lésions médullaires traumatiques (LMT) conduisent à une atteinte des voies nerveuses sensitives et motrices. Leur taux de mortalité reste très élevé, d'où la nécessité de trouver de nouveaux traitements. Les Cellules Gliales Olfactives (CGOs) représentent un candidat intéressant de par leur fonction au sein du système olfactif primaire. La découverte d'une population de cellule souche neurale bordant le canal central de la moelle spinale (MS) adulte, appelées cellules épendymaires, suscite un nouvel espoir dans le domaine des biothérapies. Ce travail de thèse a permis d'étudier l'effet d'une transplantation de CGOs sue le comportement des cellules résidentes de la moelle spinale et notamment les cellules souches épendymaires qui, en association avec les astrocytes et les péricytes, participent aux mécanismes de guérison des LMT. L'utilisation du modèle murin hFoxJ1-CreERT2::YFP (permettant le suivi spécifique des cellules épendymaires et de leur progénie), a montré que les CGOs augmentaient in vitro le potentiel d'auto-renouvellement des cellules souches de la MS et modifiaient leur voie de différenciation vers un type neural. In vivo, la transplantation de CGOs augmente la prolifération des cellules épendymaires ainsi que leur différenciation en astrocytes hypo-réactifs conduisant à la formation d'un environnement post-lésionnel bénéfique à la survie neuronal et l'établissement d'une neurogenèse. Nos travaux ont montré pour la première fois que la transplantation de CGOs après LMT permettait la génération de nouveaux neurones. Ceci constitue un nouvel espoir dans l'établissement de stratégies thérapeutiques pour le traitement des LMT chez l'Homme. / The spinal cord injuries (SCI) lead to the damages of the spinal cord or nerves and often cause permanent changes in body functions leading to the death. Cell therapies have raised great hope for regenerative medicine. Clinical data showed that the olfactory ensheathing cells (OECs) enhanced functional recovery after SCI and could be a very attractive therapeutic approach. Moreover, the discovery of a new endogenous resident stem cell population, lining the central canal of the spinal cord, named ependymal stem cells, represents a new hope for the therapy. This thesis analyzed the role of OECs transplantation, on the behaviour of ependymal stem cells since these cells, together with astrocytes and pericytes significantly contribute to the recovery of SCI. The use of the mouse model hFoxJ1-CreERT2::YFP (allowing to specifically follow the ependymal stem cells ant their progeny) showed that OECs increased in vitro the self-renewal potential of spinal cord stem cells and modified their differentiation pathway towards a neural type. In vivo, OECs transplantation significantly increases the proliferation of ependymal cells and their differenciation into hypo-reactive astrocytes leading to the formation of a beneficial environment to neuronal survival and the neurogenesis establishment. Our results also showed for the first time that OECs transplantation after SCI allows the generation of new neurons by non-ependymal cell-derived progenitors. These results represent a new hope in the establishment of therapeutic strategies for the treatment of SCI in humans.

Les métalloproteases matricielles (MMPs) appartiennent à une famille d'endopéptidases dépendantes du zinc, présentent sous forme secrétée ou membranaire (MT-MMP) et qui jouent un rôle fondamental dans la signalisation cellulaire. L'activité des MMPs est régulée par leur inhibiteurs endogènes, les inhibiteurs tissulaires des MMPs (TIMPs). Le système MMP/TIMP régule les interactions cellule-cellule et cellule-matrice extra cellulaire et module la motilité cellulaire par clivage protéolytique des composants de la matrice extra cellulaire aussi bien lors de processus physiologiques que dans des situations pathologiques.Dans un premier temps, nous avons mis en évidence le rôle de TIMP-1 dans la modulation de la croissance neuritique et la morphologie neuronale, via l'inhibition de MMP-2 et non de MMP-9. souches de la muqueuse olfactive (OE-MSCs). Nous montrons dans cette étude que les gélatinases MMP-2 et MMP-9 ainsi que la MMP membranaire MT1-MMP, sont impliquées dans la migration des OE-MSCs. Nous montrons également que les gélatinases sont probablement impliquées dans les propriétés neurotrophiques des OE-MSCs et des cellules engainantes olfactives.L'ensemble de ces résultats apporte de nouveaux éléments fondamentaux, dans la compréhension du rôle du système MMP/TIMP dans les processus post-lésionnels qui ont lieu au sein du système nerveux central. / The matrix metalloproteinases (MMPs) belong to a growing family of Zn2+-dependent endopeptidases, secreted or membrane-bound (MT-MMP), which play a fundamental role in the cell signalling. The activity of the MMPs is regulated by their endogenous inhibitors, the tissue inhibitors of MMPs (TIMPs). The MMP / TIMP system regulates the cell-cell and cell-extracellular matrix interactions and modulates the cellular motility through the cleavage of protein components of the extracellular matrix, as well during physiological and pathological conditions.Our results suggest that TIMP-1 is implicated in the modulation of the neurite outgrowth and morphology of cortical neurons through the inhibition at least in part, of MMP-2 and not MMP-9. Afterward, we study of the system MMP / TIMP in the migration of the stem cells of olfactory ectomesenchymal stem cells (OE-MSCs). We show that gelatinases MMP-2 and MMP-9 as well as MT1-MMP, are involved in OE-MSCs migration. We also show that gelatinases are probably involved in neurotrophic properties of the OE-MSCs and olfactory ensheathing cells.Altogether, these results provide new evidences on the role of MMP/TIMP system in central nervous system post-lesional processes.

Lorsque le système nerveux central des mammifères est lésé, un ensemble de réactions secondaires impliquant l’inflammation et une gliose réactive conduit à la formation d’une cicatrice gliale qui inhibe la régénération axonale. Dans le cas d’une lésion de la moelle épinière l’absence de réparation efficace des réseaux axonaux lésés peut conduire à la paraplégie ou à la tétraplégie. Aujourd’hui on estime à plus de 2,5 millions le nombre d’individus dans le monde souffrant de ces handicaps et il n’existe à ce jour aucun traitement validé pour améliorer la situation des patients. Cependant, certaines approches de thérapie moléculaire, cellulaire, et de réadaptation semblent toutefois prometteuses sur modèle animal. La dégradation des chondroitines sulfates protéoglycanes (CSPGs), principales protéines inhibitrices de la cicatrice gliale, par clivage des coeurs protéiques et ou des chaînes latérales glycosaminoglycanes favorise la régénération axonale et entraîne une récupération fonctionnelle. Des études ont montré que la métalloprotéase matricielle MMP‐2 est capable de dégrader le coeur protéique de ces CSPGs. Par ailleurs, les cellules engainantes de la muqueuse olfactive (CEOs) occupent une place privilégiée parmi les types cellulaires proposés dans la thérapie cellulaire en favorisant la croissance axonale et la récupérationfonctionnelle après lésion de la moelle épinière. Cependant, les mécanismes qui sous‐tendent les propriétés régénératrices des CEOs restent essentiellement inconnus. Dans notre Thèse, nous présentons nos travaux en trois parties. Dans la première, nous montrons in vitro que : i) les CEOs en culture primaire secrètent des taux élevés de MMP‐2, au moins en partie active ; ii) les gélatinases MMP‐2 et MMP‐9 présentent une sécrétion vésiculaire golgi‐dépendante; iii) la distribution des vésicules contenant les MMPs est liée à celle du cytosquelette et des moteurs moléculaires qui participent probablement à une sécrétion focalisée de ces molécules en fonction d’interactions entre le milieu extracellulaire et le cytosquelette ; iv) les MMPs peuvent avoir une distribution nucléaire dans les CEOs ; v) MMP‐2 jouerait un rôle dans la migration des CEOs, un processus important dans leurs capacités à réparer le tissu nerveux. Dans la seconde partie de notre thèse, nous avons développé un modèle de cicatrice gliale in vitro et nous montrons que : i) la migration des cellules astrocytaires de la cicatrice gliale in vitro est sensible aux effets des inhibiteurs des MMPs, contrairement aux cellules microgliales ; ii) les CEOs lèvent l’inhibition de croissance axonale due aux cellules astro‐microgiales ; iii) le potentiel des CEOs à créer un environnement permissif à la croissance axonale serait lié aux gélatinases sécrétées par ces cellules, en particulier MMP‐2. Dans la troisième partie de notre Thèse, nous avons évalué in vivo si MMP‐2 contribuait aux effets bénéfiques des CEOs. Nous montrons pour la première fois, dans un model animal d’hémisection de la moelle épinière, et en utilisant des approches anatomiques, électrophysiologiques et d’analyse de la locomotion, qu’une administration chronique de MMP‐2 recombinante : i) augmente le nombre et le diamètre des axones du coté distal du site de lésion ; ii) restaure la réponse évoquée du reflexe‐H distal au site de lésion ; iii) améliore la réponse respiratoire à la fatigue musculaire induite électriquement et, iv) le plus important, améliore la récupération de la locomotion. L’ensemble de notre travail suggère que MMP‐2 sécrétée par les CEOs jouerait un rôle important des les propriétés bénéfiques de ces cellules lorsqu’elles sont transplantées dans des sites de lésions de la ME, et que cette MMP présente un réel potentiel thérapeutique qui reste à explorer. / When the mammalian central nervous system is injured, a set of secondary reactions involving inflammation and reactive gliosis leads to the formation of a glial scar that inhibits axonal regeneration. In the case of a spinal cord lesion, the lack of effective repair of injured axonal networks can lead to paraplegia or quadriplegia. Today it is estimated that more than 2.5 million people are suffering from these handicaps worldwide, and there is as yet no validated treatment to improve the situation of patients. However, based on animal models, some molecular, cellular, and rehabilitation therapy approaches seem promising. Degradation of chondroitin sulfate proteoglycan (CSPG), the main inhibitory protein of the glial scar, by cleavage of either the protein core or side chains glycosaminoglycans, promotes axonal regeneration and leads to functional recovery. Studies have shown that the matrix metalloproteinase MMP-2 is capable of degrading the core protein of the CSPG. In addition, olfactory mucosa ensheathing cells (OECs) represent the most promising cell type for promoting axonal growth and functional recovery after spinal cord injury. However, the mechanisms underlying the regenerative properties of OECs remain essentially unknown. Here, we present our work in 2 parts. First, we show in vitro that: i) OECs in primary culture secrete high levels of active MMP-2; ii) both gelatinases, MMP-2 and MMP-9, have a vesicular Golgi-dependent secretion; iii) the distribution of vesicles containing the MMPs is linked to cytoskeleton and molecular motors distribution, which are probably involved in focused secretion of these molecules; iv) MMPs may have a nuclear distribution in OECs; v) MMP-2 plays a role in the migration of EOCs, an important process in their ability to repair nerve tissue. In the second part of my work, we evaluated whether the MMP-2 contributed to the beneficial effects of EOCs. We used an in vivo approach and we show for the first time, in an animal model of hemisection of the spinal cord, and using anatomical, electrophysiological analysis of locomotion approaches, that a chronic administration of recombinant MMP-2: i) increases the number and diameter of axons in the distal side of the site of injury; ii) restores the response-evoked H-reflex distal to the lesion site, iii) enhances the respiratory response to electrically-induced muscle fatigue, and iv) most importantly, improves the recovery of locomotion. All our work suggests that MMP-2, secreted by the EOCs, plays an important role in the recovery properties of these cells, when transplanted into spinal cord lesions, and that this MMP has a real therapeutic potential that remains to be explored.